Gene Clone and Characterization of a Novel Thermostable β-Galactosidase with Transglycosylation Activity from Thermotoga naphthophila RUK-10

doi:10.1007/s40242-015-5032-3

Chemical Research in Chinese Universities ›› 2015, Vol. 31 ›› Issue (4): 564-568.doi: 10.1007/s40242-015-5032-3

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Gene Clone and Characterization of a Novel Thermostable β-Galactosidase with Transglycosylation Activity from Thermotoga naphthophila RUK-10

YANG Jingwen, DI Xiangjun, WANG Man, GAO Renjun

Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, School of Life Science, Jilin University, Changchun 130012, P. R. China

Received:2015-01-23 Revised:2015-04-21 Online:2015-08-01 Published:2015-05-11
Contact: GAO Renjun E-mail:gaorj@jlu.edu.cn
Supported by:
Supported by the National High Technology Research and Development Program(“863” Program) of China(No. 2013AA102104).

Abstract

Abstract:

We cloned and expressed a new recombinant β-galactosidase(TN0949) from Thermotoga naphthophila RKU-10 with the pET28a(+) vector system in Escherichia coli BL21(DE3), and determined its catalytic capability to synthesize alkyl glucosides. The recombinant enzyme was purified to a single band via heat treatment and Ni²⁺-NTA affinity chromatography. The molecular mass of the recombinant enzyme was estimated to be 79 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). TN0949 can hydrolyze o-nitrophenyl β-D- galactopyranoside at the optimum pH and temperature of 6.5 and 80 ℃, respectively. TN0949 can also hydrolyze lactose at the optimum pH and temperature of 5.2 and 80 ℃, respectively. The K_m values for the hydrolyses of o-nitrophenyl β-D-galactopyranoside and lactose were 0.82 and 83.65 mmol/L, respectively. TN0949 was stable over a wide range of pH(3.0 to 7.0) after 24 h of incubation. The half-lives of TN0949 at 75, 80 and 85 ℃ were 22, 6 and 1.33 h, respectively. The enzyme displayed the capability to use lactose as the transglycosylation substrate to synthesize butyl galactopyranoside and hexyl galactopyranoside, indicating its suitability as a candidate industrial biocatalyst.

Key words: β-Galactosidase, Thermostable, Transglycosylation

YANG Jingwen, DI Xiangjun, WANG Man, GAO Renjun. Gene Clone and Characterization of a Novel Thermostable β-Galactosidase with Transglycosylation Activity from Thermotoga naphthophila RUK-10[J]. Chemical Research in Chinese Universities, 2015, 31(4): 564-568.

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Gene Clone and Characterization of a Novel Thermostable β-Galactosidase with Transglycosylation Activity from Thermotoga naphthophila RUK-10

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[1]	KONG Fansi, YANG Jingwen, ZHEN Zhen, LIANG Tingting, ZHU Dongliang, GAO Renjun, XIE Guiqiu. Gene Cloning and Molecular Characterization of a β-Glucosidase from Thermotoga Naphthophila RUK-10: an Effective Tool for Synthesis of Galacto-oligosaccharide and Alkyl Galactopyranosides [J]. Chemical Research in Chinese Universities, 2015, 31(5): 774-780.
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